Evaluation of a Functional Assay for Radiosensitivity in the Pediatric Prospective Cohort ARPEGE

Purpose/Objective(s)Approximately 900 children/adolescents are treated with radiotherapy (RT) every year in France. However, among the 80% of survivors, the cumulative incidence of long-term morbidity – including second malignancies - reach 73.4% thirty years after the cancer diagnosis. Identifying a priori the subjects at risk for RT sequelae is a major challenge of pediatric oncology. Individual radiosensitivity (IRS) of children/adolescents is unknown at this time. The cellular response to radiation depends on the recognition and repair of radiation-induced DNA double-strand breaks (DSB). Unrepaired DSB as well as a delay in the intra nuclear activation of the pATM protein are common features to patients with RT toxicity. From these conclusions, we proposed and retrospectively validated a functional IRS immunofluorescence assay based on Radio-Induced ATM Nucleoshuttling (RIANS) (Granzotto et al, 2016). The purpose of this study was to define prospectively the predictive impact of RIANS assay in children.Materials/MethodsPatients of the ARPEGE prospective open-label, non-randomized multicenter clinical trial were included in the study and prospectively evaluated for early toxicities. For each patient, skin samples were collected prior RT to raise a primary dermal fibroblast line and carry out in blind the RIANS assay. Different endpoints were measured: residual double-strand breaks at 24 h (γH2AX marker), pATM foci at 10 min and 1 h (pATM marker) and micronuclei at 24 h respectively after ex vivo radiation (2 Gy). The procedures were repeated in triplicates in blind in two independent expert centers (Neolys and Institut de Cancerologie de Lorraine). In parallel early toxicity was reported according to NCI-CTCAE v4.0 three months after the completion of RT. Patients with grade <2 or ≥2 toxicity were considered as radioresistant (RR) and radiosensitive (RS) respectively. We correlated the parameters of the RIANS assay according to toxicity with logistic regression.ResultsAmong the 29 analyzable patients, 37 % exhibit brain cancers, 10% Hodgkin lymphomas, 10% nephroblastoma, 10% neuroblastoma, 10% Ewing cancer and 13% malignant mesenchymal cancer. The mean delivered dose was 32.5 Gy (range 1,5 - 60 Gy) with fractionation between 1.5 to 2 Gy. 16/29 (56%) patients were clinically considered as RS. There was no significant difference in total dose delivered between the two groups. Among the radiobiological factors tested, the number of Micronuclei at 24h post irradiation provided the best discrimination among RS patients (p-value= 0.0216). Overall, the RIANS assay provided the following performance: sensitivity=0.82, specificity=0.70 and AUC=0.75 (0.550-0.888).ConclusionARPEGE is the first study to document the distribution of IRS in such a population. Screening hypersensitive patients would be a major step forward in the management of cancers, opening a way to personalized pediatric oncology. Approximately 900 children/adolescents are treated with radiotherapy (RT) every year in France. However, among the 80% of survivors, the cumulative incidence of long-term morbidity – including second malignancies - reach 73.4% thirty years after the cancer diagnosis. Identifying a priori the subjects at risk for RT sequelae is a major challenge of pediatric oncology. Individual radiosensitivity (IRS) of children/adolescents is unknown at this time. The cellular response to radiation depends on the recognition and repair of radiation-induced DNA double-strand breaks (DSB). Unrepaired DSB as well as a delay in the intra nuclear activation of the pATM protein are common features to patients with RT toxicity. From these conclusions, we proposed and retrospectively validated a functional IRS immunofluorescence assay based on Radio-Induced ATM Nucleoshuttling (RIANS) (Granzotto et al, 2016). The purpose of this study was to define prospectively the predictive impact of RIANS assay in children. Patients of the ARPEGE prospective open-label, non-randomized multicenter clinical trial were included in the study and prospectively evaluated for early toxicities. For each patient, skin samples were collected prior RT to raise a primary dermal fibroblast line and carry out in blind the RIANS assay. Different endpoints were measured: residual double-strand breaks at 24 h (γH2AX marker), pATM foci at 10 min and 1 h (pATM marker) and micronuclei at 24 h respectively after ex vivo radiation (2 Gy). The procedures were repeated in triplicates in blind in two independent expert centers (Neolys and Institut de Cancerologie de Lorraine). In parallel early toxicity was reported according to NCI-CTCAE v4.0 three months after the completion of RT. Patients with grade <2 or ≥2 toxicity were considered as radioresistant (RR) and radiosensitive (RS) respectively. We correlated the parameters of the RIANS assay according to toxicity with logistic regression. Among the 29 analyzable patients, 37 % exhibit brain cancers, 10% Hodgkin lymphomas, 10% nephroblastoma, 10% neuroblastoma, 10% Ewing cancer and 13% malignant mesenchymal cancer. The mean delivered dose was 32.5 Gy (range 1,5 - 60 Gy) with fractionation between 1.5 to 2 Gy. 16/29 (56%) patients were clinically considered as RS. There was no significant difference in total dose delivered between the two groups. Among the radiobiological factors tested, the number of Micronuclei at 24h post irradiation provided the best discrimination among RS patients (p-value= 0.0216). Overall, the RIANS assay provided the following performance: sensitivity=0.82, specificity=0.70 and AUC=0.75 (0.550-0.888). ARPEGE is the first study to document the distribution of IRS in such a population. Screening hypersensitive patients would be a major step forward in the management of cancers, opening a way to personalized pediatric oncology.